Skin is composed of a thin outer layer, the epidermis, and a thicker inner layer, the dermis, which are separated by the basement membrane. Subcutaneous tissue is located beneath the dermis, which contains mostly fat, and beneath that fasciae and muscles are found. The epidermis forms a tough, waterproof protective coating that contains dead cells on its outside surface and living cells on its inner side. The living cells replace the dead cells that are continually worn away. The dermis is a living tissue containing blood vessels, nerves, sweat glands and hair follicles.
Collagen is the main protein of connective tissue in animals, and its fibrillar form gives skin its mechanical strength. Each collagen fiber is composed of three long; helical polypeptide chains that bind tightly to each other. Synthesis and breakdown of collagen is indicative of skin integrity as well as of various skin diseases and disorders.
Collagen type I is synthesized as procollagen with a small amino terminal and a larger carboxy terminal propeptide, respectively termed procollagen type I amino terminal peptide (PINP) and type I procollagen carboxy terminal peptide (PICP). Once secreted into the extracellular space, the propeptides are removed by specific endopeptidases, thus allowing integration of the rigid collagen triple helix into the growing fibril (Miyahara et al. (1982) J Biol Chem. 257:8442-8448). The 100 kD PICP formed during this process is released into the blood. A stoichiometric ratio of 1:1 exists between the number of collagen molecules produced and that of PICP released.12 Therefore, the serum concentration of PICP has been proposed as a marker of collagen type I synthesis (Parfitt et al. (1987) J Bone Miner Res. 2:427-436).
Collagen type I carboxy terminal cross-linked telopeptide (abbreviated as CTX-I or CITP) is a 12 kD peptide produced, together with other peptides, when collagen fibrils undergo resorption (Alexander et al. Extracellular matrix degradation. In: Hay ed. Cell Biology of Extracellular Matrix. 2nd ed. New York, N.Y.: Plenum Press pp. 255-275). CITP is constituted by the carboxy terminal telopeptide (CTP) parts of two α1 chains of one collagen molecule and one α1 or α2-derived helical chain of another collagen molecule, cross-linked by a pyridinoline ring. This peptide is found in an immunochemically intact form in blood, where it appears to be derived from tissues (Risteli et al. (1993) Clin Chem. 39:635-640).
Osteoporosis, characterized by a decrease in bone mass, is associated with collagen degradation, specifically degradation of type I collagen which is the major organic component in the bone. Direct estimation of type I collagen degradation can be made by measuring the amount of type I collagen carboxy terminal telopeptide (C-telopeptide, CTP-I), or the amount of type I collagen amino terminal telopeptide (N-telopeptide, NTP-I), which are non-triple helical extension peptides found respectively at the C-terminal and the N-terminal ends of the collagen fiber.
For example, U.S. Pat. No. 5,538,853 discloses the use of antibody raised against type I collagen carboxy terminal cross-linked telopeptide isolated from decalcified human or animal bone for determining the concentration of liberated CTP in a sample, particularly serum or urine, to assess the degradation of type I collagen. Kits for bone degradation assays based on this disclosure are commercially available (Orion Diagnostica, Espoo, Finland).
U.S. Pat. No. 5,753,450 discloses assays for monitoring the levels of the C-telopeptide of human type I collagen as a marker for collagen degradation and specific indicator of bone resorption.
Pyridinoline and deoxypyridinoline collagen cross-link molecules also used as bone resorption markers. The relative levels of pyridinoline and deoxypyridinoline in urine from patients with rheumatoid arthritis has been disclosed as a marker of synovial tissue collagen degradation for that disease (Kaufmann J. et al. (2003) Rheumatology (Oxford) 42(2):314-20).
Serum levels of type I procollagen carboxy terminal propeptide (PICP) have been disclosed to be indicative of the clinical course of scirrhous carcinoma of the stomach (Kohda et al. (1991) Gut 32(6):624-629), and of myocardial fibrosis in hypertensive heart disease (Querejeta et al. (2000) Circulation 101:1729).
U.S. Pat. No. 6,916,604 discloses a method of assaying type I collagen fragments in a body fluid sample, comprising contacting the body fluid with a synthetic type I collagen N-telopeptide sequence, and an antibody immunoreactive with said sequence, using a competitive binding assay.
U.S. Pat. No. 6,509,450 discloses an immunoassay kit for the quantification of degradation products of carboxy-terminal telopeptides of type I collagen in a human serum sample.
U.S. Pat. No. 6,204,367 discloses an enzyme linked immunosorbent assay kit for the quantification of degradation products of carboxy-terminal telopeptides of type I collagen in a human serum sample.
U.S. Pat. No. 6,153,732 discloses a kit for detecting an analyte indicative of type II collagen resorption in vivo.
Basement membrane is a highly specialized part of the extracellular matrix that forms thin sheets that separate the cells of organs from the fibrillar connective tissues. The basement membrane is composed of several proteins, many of which are found only in these structures. Type IV collagen is the major structural component but other specific protein components include laminin, entactin (nidogen), perlecan, α6-β4-integrin and proteoglycans. Additionally, the basement membrane may contain fibronectin and type VII collagen, that are also present in other extracellular matrices. It is currently believed that there are several proteins that are specifically found in basement membrane of certain tissues. For example, the protein known as pemphigoid antigen appears to be limited to the basement membrane of skin.
An assay for detecting serum levels of collagen IV and laminin and use thereof for monitoring liver diseases has been disclosed (Bolarin et al (2007) Nig. Q. J. Hosp. 17(1):42-52).
Heating of the skin, however brief, can cause damage to the cells of its living tissue. Such damage typically is referred to as a burn. Generally, skin burns are categorized into degrees that indicate the depth of the burn injury. First degree burns cause redness of the skin and affect only the epidermis. Such burns heal quickly, but the damaged skin may peel away after a day or two. “Sunburn” is an example of a first degree burn. A second degree burn damages the skin more deeply, extending into the dermis and usually causes blistering. However, some of the dermis is left to recover. A third degree burn destroys the full thickness of the skin. In fourth degree burn, the burn extends beyond the skin to the fat, muscle and bone.
Experience has shown that second degree superficial dermal (or partial-thickness) burns will normally heal spontaneously within two weeks with minimal scarring, whereas deep dermal second degree and third degree (or full-thickness) burns usually result in necrotic skin and in very slow wound healing.
It is of major clinical importance to diagnose the depth of the burn as early as possible in order to determine the optimal course of patient treatment. However, it is usually almost impossible to assess the exact degree of the burn in the first 48-72 hours after the burn has been sustained.
Several methods have been proposed for evaluating burn injury, particularly to distinguish necrotic tissue from viable tissue. Clinical criteria utilized to distinguish burn depth include sensitivity to pin prick, visual appearance, and viable cutaneous circulation, but these criteria are not objective and they do not constitute a practical method for evaluating burn injury. Imaging techniques such as passive infrared thermography, laser Doppler, false-positive images, and high-frequency ultrasound are expensive and not sufficiently informative. Intravenous administration of fluorescent drugs such as indocyanine green and fluorescein, followed by detection with a fiber-optic instrument, has been attempted with limited success. Such techniques are hampered by the period of time required for the fluorescent drugs to reach the burn site and/or the time required for their clearance from the burn site, thus limiting the frequency of testing. Further, intravenous administration is counter-indicated in many burn cases since burn patients frequently suffer from vasoconstriction at the early stages.
The prior art does not teach or suggest that measurement of collagen or basement membrane degradation products may be used for the evaluation of burn severity.
There remains an unmet need for accurate, rapid and low cost methods and kits for evaluating the severity of a burn injury.